鸭源新型鹅细小病毒抗体间接ELISA检测方法的建立及应用

Establishment and application of indirect ELISA method for detection of antibodies for a novel goose parvovirus from ducks

新型鹅细小病毒(novel goose parvovirus,NGPV)是由鹅细小病毒(goose parvovirus,GPV)变异而来,可感染雏鸭,引起以鸭短喙侏儒综合征为主要特征的传染性疾病。2015年该病在我国鸭群中大暴发,严重影响鸭出栏率,给我国的养殖行业带来巨大的经济损失。为建立一种鸭源NGPV抗体的间接ELISA检测方法,用于鸭感染细小病毒的血清学检测和鸭免疫细小病毒疫苗后抗体水平监测,本研究利用生物信息学预测了NGPV YICH株的结构蛋白VP3的抗原表位,选择了抗原富集区域(aa250~525)进行原核表达,经蛋白纯化获得了可溶性的VP3截短蛋白VP3-tr。以VP3-tr为包被抗原,经过棋盘滴定方法优化各个反应条件,建立了NGPV抗体的间接ELISA检测方法。利用优化后的间接ELISA方法对30份GPV阴性鸭血清进行检测,确定阴阳性临界值为0.219。ELISA方法的灵敏性、特异性和重复性检测结果表明,该方法灵敏度较高,特异性良好,不与鸭坦布苏病毒、新城疫病毒、禽流感病毒、鸭疫里默杆菌、大肠杆菌病原阳性血清发生交叉反应;批内和批间重复试验的变异系数均小于6%,重复性良好。用该方法与Western blot检测方法分别对147份临床血清样品进行检测,结果表明ELISA检测阳性率为85.0%,Western blot检测率为69.4%,两者符合率为84.4%。综上所述,本研究成功建立了鸭源NGPV抗体的间接ELISA检测方法,为鸭感染NGPV后的感染情况监测以及疫苗免疫评估提供了更简便可靠的方法。

Novel goose parvovirus(NGPV)is a variant of goose parvovirus(GPV),which can infect young ducks and cause an infectious disease characterized by duck short-beaks and dwarf syndrome.In 2015,the disease broke out in duck herd in China,which seriously affected the emergence rate of ducks and brought huge economic losses to breeding industry of China.To establish an indirect ELISA method for the detection of antibodies against NGPV for the serological detection of parvovirus infection in ducks and the monitoring of antibody levels in ducks immunized with parvovirus vaccine,the antigenic epitope of the structural protein VP3 of NGPV YICH strain was predicted by bioinformatics,and the antigen rich region(250-525 aa)was selected for prokaryotic expression.After protein purification,the soluble truncated protein VP3-tr was obtained.Then,using VP3-tr as coating antigen,a new indirect ELISA method for detection of NGPV antibody was established by optimizing the reaction conditions through checkerboard titration method.The optimized indirect ELISA method was used to detect 30 duck serum samples negative for GPV,and the cut-off value of negative and positive was O.219.The sensitivity,specificity and repeatability of ELISA showed that the method had high sensitivity and good specificity,and did not cross react with the positive serums of duck Tambusu virus,Newcastle disease virus,avian influenza virus,Riemerella anatipestifer and E.coil.The coefficients of variation of intra-and inter-assay repeated tests were less than 6%,which showed good reproducibility.The results showed that the positive rate of ELISA was 85.0%,and that of Western blot was 69.4%.The coincidence rate of ELISA and Western blot was 84.4%.In conclusion,this study successfully established an indirect ELISA method for the detection of antibodies to the NGPV from duck,which provides a simple and reliable method for the surveillance of ducks infected with NGPV and the evaluation of vaccine immunity.